幹細胞的標誌Nestin，為中間絲蛋白家族成員之一。其對於細胞完整性，遷移以及分化扮演著一個重要的角色。Nestin高表現在三分之一的胰臟癌病人中，且其陽性表現關係到腫瘤分期和胰臟周圍侵襲的現象。然而，胰臟癌中，Nestin的功能仍尚未明瞭。我們的研究指出:在胰臟癌細胞中過度表達Nestin可以增加細胞移動的能力以及驅使上皮-間質轉化的表現型出現；相反地，我們抑制內生型的Nestin表現，則會降低轉移率並使更多細胞回復成上皮細胞的型態。此外，在異種移植NOD/SCID小鼠體內實驗中，Nestin被抑制之後，會減少腫瘤發生且降低腫瘤生成的體積。有趣的是，我們觀察到在胰臟癌中，Nestin的表現與Smad4的存在有關。這個現象指出，在胰臟癌中，Nestin的表現可能是被TGF-b1/SMAD4這條訊息路徑所調控。為了確認此現象，我們選擇人類胰臟癌細胞PANC-1，以及PANC-1 shSmad4來做實驗。我們將兩種細胞加入了TGF-b1，去分析Nestin的表現是否有所差異。我們的結果指出，在胰臟癌中，TGF-b1/SMAD會藉由Smad4去誘導Nestin蛋白表現。而我們更近一步地闡述，有兩種中間絲蛋白的抑制劑，CD和WFA，具有抗Nestin活性的能力。我們認為這些抑制劑具有作為抗轉移地藥物的潛力。總而言之，我們的研究指出Nestin在TGF-b1所誘導出的EMT現象中，其所扮演的一個重要的角色，且將這個發現應用在臨床中。我們認為對抗Nestin的治療策略，未來可應用在胰臟癌的抗轉移療法。

Stem cell marker Nestin is an intermediate filament protein that plays an important role in cell integrity, migration and differentiation. Nestin expression occurs in approximately one-third of pancreatic ductal adenocarcinoma (PDAC) cases, and its expression positively correlates with tumor stage and peripancreatic invasion. Little is known of the mechanisms by which Nestin influences PDAC progression. We showed that Nestin overexpression in PDAC cells increased cell motility and drove phenotypic changes associated with the epithelial-mesenchymal transition in vitro, conversely, knockdown of endogenous Nestin expression reduced the migration rate and cells reverted to a more epithelial phenotype. In vivo mice studies showed that knockdown of Nestin significantly reduced tumor incidence and volume in xenografts. Expression of the Nestin protein was associated with Smad4 status in PDAC cells, hence Nestin expression might be regulated by the TGF-b1/SMAD4 pathway in PDAC. We examined Nestin expression after TGF-b1 treatment in human pancreatic cancer PANC-1, and PANC-1 shSmad4 cells. The TGF-b/SMAD pathway induced Nestin protein expression in PDAC cells through Smad4 in a dependent manner. Moreover, increased Nestin expression caused a positive feedback loop in the TGFb/SMAD signaling system.
Finally, we demonstrated that 2 anti-microtubule inhibitors, Cytochalasin D (CD) and Withaferin A (WFA), exhibited anti-Nestin activity; these inhibitors might be potential anti-metastatic drugs. Our findings uncovered a novel role of Nestin in regulating TGF-b1-induced EMT. Anti-Nestin therapeutics are under development as a potential treatment for PDAC metastasis.

Contents
Abstract in Chinese ------------------------------------------------- ii
Abstract in English -------------------------------------------------- iii
Introduction ------------------------------------------------------------ 1
Materials and Methods --------------------------------------------- 6
Results ---------------------------------------------------------------- 15
Discussion ------------------------------------------------------------ 25
Figures and Tables ------------------------------------------------ 31
References ----------------------------------------------------------- 60

1. Magee CJ, Ghaneh P, Hartley M, Sutton R, Neoptolemos JP. The role of adjuvant therapy for pancreatic cancer. Expert Opin Investig Drugs 2002;11(1):87-107.
2. Takeda S, Inoue S, Kaneko T, Harada A, Nakao A. The role of adjuvant therapy for pancreatic cancer. Hepatogastroenterology 2001;48(40):953-6.
3. Strimpakos A, Saif MW, Syrigos KN. Pancreatic cancer: from molecular pathogenesis to targeted therapy. Cancer Metastasis Rev 2008;27(3):495-522.
4. Jamieson NB, Foulis AK, Oien KA, et al. Peripancreatic fat invasion is an independent predictor of poor outcome following pancreaticoduodenectomy for pancreatic ductal adenocarcinoma. J Gastrointest Surg;15(3):512-24.
5. Sergeant G, Ectors N, Fieuws S, Aerts R, Topal B. Prognostic relevance of extracapsular lymph node involvement in pancreatic ductal adenocarcinoma. Ann Surg Oncol 2009;16(11):3070-9.
6. Kawamoto M, Ishiwata T, Cho K, et al. Nestin expression correlates with nerve and retroperitoneal tissue invasion in pancreatic cancer. Hum Pathol 2009;40(2):189-98.
7. Bozzuto G, Ruggieri P, Molinari A. Molecular aspects of tumor cell migration and invasion. Ann Ist Super Sanita;46(1):66-80.
8. Steeg PS. Tumor metastasis: mechanistic insights and clinical challenges. Nat Med 2006;12(8):895-904.
9. Zetter BR. Angiogenesis and tumor metastasis. Annu Rev Med 1998;49:407-24.
10. Netherton SJ, Bonni S. Suppression of TGFbeta-induced epithelial-mesenchymal transition like phenotype by a PIAS1 regulated sumoylation pathway in NMuMG epithelial cells. PLoS One;5(11):e13971.
11. Smit MA, Peeper DS. Epithelial-mesenchymal transition and senescence: two cancer-related processes are crossing paths. Aging (Albany NY);2(10):735-41.
12. Wu Y, Zhou BP. New insights of epithelial-mesenchymal transition in cancer metastasis. Acta Biochim Biophys Sin (Shanghai) 2008;40(7):643-50.
13. Huber MA, Kraut N, Beug H. Molecular requirements for epithelial-mesenchymal transition during tumor progression. Curr Opin Cell Biol 2005;17(5):548-58.
14. Larue L, Bellacosa A. Epithelial-mesenchymal transition in development and cancer: role of phosphatidylinositol 3' kinase/AKT pathways. Oncogene 2005;24(50):7443-54.
15. Liu Y. New insights into epithelial-mesenchymal transition in kidney fibrosis. J Am Soc Nephrol;21(2):212-22.
16. Lopez-Novoa JM, Nieto MA. Inflammation and EMT: an alliance towards organ fibrosis and cancer progression. EMBO Mol Med 2009;1(6-7):303-14.
17. Willis BC, Borok Z. TGF-beta-induced EMT: mechanisms and implications for fibrotic lung disease. Am J Physiol Lung Cell Mol Physiol 2007;293(3):L525-34.
18. Ungefroren H, Groth S, Sebens S, Lehnert H, Gieseler F, Fandrich F. Differential roles of Smad2 and Smad3 in the regulation of TGF-beta1-mediated growth inhibition and cell migration in pancreatic ductal adenocarcinoma cells: control by Rac1. Mol Cancer;2011;10:67.
19. Freeman JW, DeArmond D, Lake M, Huang W, Venkatasubbarao K, Zhao S. Alterations of cell signaling pathways in pancreatic cancer. Front Biosci 2004;9:1889-98.
20. Ottenhof NA, de Wilde RF, Maitra A, Hruban RH, Offerhaus GJ. Molecular characteristics of pancreatic ductal adenocarcinoma. Patholog Res Int;2011:620601.
21. Schniewind B, Groth S, Sebens Muerkoster S, et al. Dissecting the role of TGF-beta type I receptor/ALK5 in pancreatic ductal adenocarcinoma: Smad activation is crucial for both the tumor suppressive and prometastatic function. Oncogene 2007;26(33):4850-62.
22. Nagaraj NS, Datta PK. Targeting the transforming growth factor-beta signaling pathway in human cancer. Expert Opin Investig Drugs;2010;19(1):77-91.
23. Saitoh M, Miyazawa K. Transcriptional and post-transcriptional regulation in TGF-beta-mediated epithelial-mesenchymal transition. J Biochem ; 2012; published on line..
24. Santibanez JF, Quintanilla M, Bernabeu C. TGF-beta/TGF-beta receptor system and its role in physiological and pathological conditions. Clin Sci (Lond);2011;121(6):233-51.
25. Christiansen JJ, Rajasekaran AK. Reassessing epithelial to mesenchymal transition as a prerequisite for carcinoma invasion and metastasis. Cancer Res 2006;66(17):8319-26.
26. Tomaskovic-Crook E, Thompson EW, Thiery JP. Epithelial to mesenchymal transition and breast cancer. Breast Cancer Res 2009;11(6):213.
27. Guarino M, Rubino B, Ballabio G. The role of epithelial-mesenchymal transition in cancer pathology. Pathology 2007;39(3):305-18.
28. Small JV, Rottner K, Kaverina I. Functional design in the actin cytoskeleton. Curr Opin Cell Biol 1999;11(1):54
29. Steinert PM, Bale SJ. Genetic skin diseases caused by mutations in keratin intermediate filaments. Trends Genet 1993;9(8):280-4.
30. Wu C, Keightley SY, Leung-Hagesteijn C, et al. Integrin-linked protein kinase regulates fibronectin matrix assembly, E-cadherin expression, and tumorigenicity. J Biol
Chem 1998;273(1):528-36.
31. Kim S, Coulombe PA. Emerging role for the cytoskeleton as an organizer and regulator of translation. Nat Rev Mol Cell Biol;2010;11(1):75-81.
32. Harold FM. Molecules into cells: specifying spatial architecture. Microbiol Mol Biol Rev 2005;69(4):544-64.
33. Kim S, Coulombe PA. Intermediate filament scaffolds fulfill mechanical, organizational, and signaling functions in the cytoplasm. Genes Dev 2007;21(13):1581-97.
34. Bardeesy N, Cheng KH, Berger JH, et al. Smad4 is dispensable for normal pancreas development yet critical in progression and tumor biology of pancreas cancer. Genes Dev 2006;20(22):3130-46.
35. Quinlan R, Hutchison C, Lane B. Intermediate filament proteins. Protein Profile 1994;1(8):779-911.
36. Kreplak L, Doucet J, Dumas P, Briki F. New aspects of the alpha-helix to beta-sheet transition in stretched hard alpha-keratin fibers. Biophys J 2004;87(1):640-7.
37. Faigle W, Colucci-Guyon E, Louvard D, Amigorena S, Galli T. Vimentin filaments in fibroblasts are a reservoir for SNAP23, a component of the membrane fusion machinery. Mol Biol Cell 2000;11(10):3485-94.
38. Eckes B, Dogic D, Colucci-Guyon E, et al. Impaired mechanical stability, migration and contractile capacity in vimentin-deficient fibroblasts. J Cell Sci 1998;111 ( Pt 13):1897-907.
39. Liu T, Guevara OE, Warburton RR, Hill NS, Gaestel M, Kayyali US. Regulation of vimentin intermediate filaments in endothelial cells by hypoxia. Am J Physiol Cell Physiol;2010;299(2):C363-73.
40. Helmke BP, Goldman RD, Davies PF. Rapid displacement of vimentin intermediate filaments in living endothelial cells exposed to flow. Circ Res 2000;86(7):745-
41. Homan SM, Mercurio AM, LaFlamme SE. Endothelial cells assemble two distinct alpha6beta4-containing vimentin-associated structures: roles for ligand binding and the beta4 cytoplasmic tail. J Cell Sci 1998;111 ( Pt 18):2717-28.
42. Vitadello M, Matteoli M, Gorza L. Neurofilament proteins are co-expressed with desmin in heart conduction system myocytes. J Cell Sci 1990;97 ( Pt 1):11-21.
43. Martin de las Mulas J, Espinosa de los Monteros A, Carrasco L, Sierra MA, Vos JH. Immunohistochemical distribution of vimentin, desmin, glial fibrillary acidic protein and neurofilament proteins in feline tissues. Zentralbl Veterinarmed A 1994;41(1):1-15.
44. Miettinen M, Lehto VP, Virtanen I. Presence of fibroblast-type intermediate filaments (vimentin) and absence of neurofilaments in pigmented nevi and malignant melanomas. J Cutan Pathol 1983;10(3):188-92.
45. Yan W, Cao QJ, Arenas RB, Bentley B, Shao R. GATA3 inhibits breast cancer metastasis through the reversal of epithelial-mesenchymal transition. J Biol Chem;285(18):14042-51.
46. Pan Y, Jing R, Pitre A, Williams BJ, Skalli O. Intermediate filament protein synemin contributes to the migratory properties of astrocytoma cells by influencing the dynamics of the actin cytoskeleton. FASEB J 2008;22(9):3196-206.
47. Pitre A, Davis N, Paul M, Orr AW, Skalli O. Synemin promotes AKT-dependent glioblastoma cell proliferation by antagonizing PP2A. Mol Biol Cell;2012;23(7):1243-53.
48. Hijikata T, Nakamura A, Isokawa K, et al. Plectin 1 links intermediate filaments to costameric sarcolemma through beta-synemin, alpha-dystrobrevin and actin. J Cell Sci 2008;121(Pt 12):2062-74.
49. Li H, Cherukuri P, Li N, et al. Nestin is expressed in the basal/myoepithelial layer of the mammary gland and is a selective marker of basal epithelial breast tumors. Cancer Res 2007;67(2):501-10.
50. Kleeberger W, Bova GS, Nielsen ME, et al. Roles for the stem cell associated intermediate filament Nestin in prostate cancer migration and metastasis. Cancer Res 2007;67(19):9199-206.
51. Tsujimura T, Makiishi-Shimobayashi C, Lundkvist J, et al. Expression of the intermediate filament nestin in gastrointestinal stromal tumors and interstitial cells of Cajal. Am J Pathol 2001;158(3):817-23.
52. Sarlomo-Rikala M, Tsujimura T, Lendahl U, Miettinen M. Patterns of nestin and other intermediate filament expression distinguish between gastrointestinal stromal tumors, leiomyomas and schwannomas. APMIS 2002;110(6):499-507.
53. Florenes VA, Holm R, Myklebost O, Lendahl U, Fodstad O. Expression of the neuroectodermal intermediate filament nestin in human melanomas. Cancer Res 1994;54(2):354-6.
54. Ananthakrishnan R, Ehrlicher A. The forces behind cell movement. Int J Biol Sci 2007;3(5):303-17.
55. Ohike N, Sato M, Hisayuki T, et al. Immunohistochemical analysis of nestin and c-kit and their significance in pancreatic tumors. Pathol Int 2007;57(9):589-93.
56. Yang XH, Wu QL, Yu XB, et al. Nestin expression in different tumours and its relevance to malignant grade. J Clin Pathol 2008;61(4):467-73.
57. Busch T, Milena, Eiseler T, et al. Keratin 8 phosphorylation regulates keratin reorganization and migration of epithelial tumor cells. J Cell Sci.2012;125(pt9):2148-59
58. Beil M, Micoulet A, von Wichert G, et al. Sphingosylphosphorylcholine regulates keratin network architecture and visco-elastic properties of human cancer cells. Nat Cell Biol 2003;5(9):803-11.
59. Chu YW, Runyan RB, Oshima RG, Hendrix MJ. Expression of complete keratin filaments in mouse L cells augments cell migration and invasion. Proc Natl Acad Sci U S A 1993;90(9):4261-5.
60. Vicente-Manzanares M, Choi CK, Horwitz AR. Integrins in cell migration--the actin connection. J Cell Sci 2009;122(Pt 2):199-206.
61. Huttenlocher A, Ginsberg MH, Horwitz AF. Modulation of cell migration by integrin-mediated cytoskeletal linkages and ligand-binding affinity. J Cell Biol 1996;134(6):1551-62.
62. Itoh H, Nelson PR, Mureebe L, Horowitz A, Kent KC. The role of integrins in saphenous vein vascular smooth muscle cell migration. J Vasc Surg 1997;25(6):1061-9.
63. Lobert VH, Brech A, Pedersen NM, et al. Ubiquitination of alpha 5 beta 1 integrin controls fibroblast migration through lysosomal degradation of fibronectin-integrin complexes. Dev Cell;2010;19(1):148-59.
64. Cary LA, Chang JF, Guan JL. Stimulation of cell migration by overexpression of focal adhesion kinase and its association with Src and Fyn. J Cell Sci 1996;109 ( Pt 7):1787-94.
65. Han DC, Shen TL, Guan JL. Role of Grb7 targeting to focal contacts and its phosphorylation by focal adhesion kinase in regulation of cell migration. J Biol Chem 2000;275(37):28911-7.
66. Kim H, Nakamura F, Lee W, Hong C, Perez-Sala D, McCulloch CA. Regulation of cell adhesion to collagen via beta1 integrins is dependent on interactions of filamin A with vimentin and protein kinase C epsilon. Exp Cell Res;2010;316(11):1829-44.
67. Gorin MA, Pan Q. Protein kinase C epsilon: an oncogene and emerging tumor biomarker. Mol Cancer 2009;8:9.
68. Newton PM, Messing RO. The substrates and binding partners of protein kinase Cepsilon. Biochem J;2010;427(2):189-96.
69. Niki T, Pekny M, Hellemans K, et al. Class VI intermediate filament protein nestin is induced during activation of rat hepatic stellate cells. Hepatology 1999;29(2):520-7.
70. Sejersen T, Lendahl U. Transient expression of the intermediate filament nestin during skeletal muscle development. J Cell Sci 1993;106 ( Pt 4):1291-300.
71. Dahlstrand J, Collins VP, Lendahl U. Expression of the class VI intermediate filament nestin in human central nervous system tumors. Cancer Res 1992;52(19):5334-41.
72. Guerette D, Khan PA, Savard PE, Vincent M. Molecular evolution of type VI intermediate filament proteins. BMC Evol Biol 2007;7:164.
73. Wagner N, Wagner KD, Scholz H, Kirschner KM, Schedl A. Intermediate filament protein nestin is expressed in developing kidney and heart and might be regulated by the Wilms' tumor suppressor Wt1. Am J Physiol Regul Integr Comp Physiol
2006;291(3):R779-87.
74. Takahashi N, Itoh MT, Ishizuka B. Human chorionic gonadotropin induces nestin expression in endothelial cells of the ovary via vascular endothelial growth factor signaling. Endocrinology 2008;149(1):253-60.
75. Rutka JT, Ivanchuk S, Mondal S, et al. Co-expression of nestin and vimentin intermediate filaments in invasive human astrocytoma cells. Int J Dev Neurosci 1999;17(5-6):503-15.
76. Miyaguchi K. Ultrastructure of intermediate filaments of nestin- and vimentin-immunoreactive astrocytes in organotypic slice cultures of hippocampus. J Struct Biol 1997;120(1):61-8.
77. Takano T, Rutka JT, Becker LE. Overexpression of nestin and vimentin in ependymal cells in hydrocephalus. Acta Neuropathol 1996;92(1):90-7.
78. Shin TK, Lee YD, Sim KB. Embryonic intermediate filaments, nestin and vimentin, expression in the spinal cords of rats with experimental autoimmune encephalomyelitis. J Vet Sci 2003;4(1):9-13.
79. Sjoberg G, Jiang WQ, Ringertz NR, Lendahl U, Sejersen T. Colocalization of nestin and vimentin/desmin in skeletal muscle cells demonstrated by three-dimensional fluorescence digital imaging microscopy. Exp Cell Res 1994;214(2):447-58.
80. Cizkova D, Soukup T, Mokry J. Expression of nestin, desmin and vimentin in intact and regenerating muscle spindles of rat hind limb skeletal muscles. Histochem Cell Biol 2009;131(2):197-206.
81. Zou J, Yaoita E, Watanabe Y, et al. Upregulation of nestin, vimentin, and desmin in rat podocytes in response to injury. Virchows Arch 2006;448(4):485-92.
82. Chou YH, Khuon S, Herrmann H, Goldman RD. Nestin promotes the phosphorylation-dependent disassembly of vimentin intermediate filaments during mitosis. Mol Biol Cell 2003;14(4):1468-78.
83. Sahlgren CM, Mikhailov A, Vaittinen S, et al. Cdk5 regulates the organization of Nestin and its association with p35. Mol Cell Biol 2003;23(14):5090-106.
84. Sahlgren CM, Mikhailov A, Hellman J, et al. Mitotic reorganization of the intermediate filament protein nestin involves phosphorylation by cdc2 kinase. J Biol Chem 2001;276(19):16456-63.
85. Thomas PA, Kirschmann DA, Cerhan JR, et al. Association between keratin and vimentin expression, malignant phenotype, and survival in postmenopausal breast cancer patients. Clin Cancer Res 1999;5(10):2698-703.
86. LaRocca PJ, Rheinwald JG. Coexpression of simple epithelial keratins and vimentin by human mesothelium and mesothelioma in vivo and in culture. Cancer Res 1984;44(7):2991-9.
87. Wagner M, Greten FR, Weber CK, et al. A murine tumor progression model for
pancreatic cancer recapitulating the genetic alterations of the human disease. Genes Dev 2001;15(3):286-93.
88. Schussler MH, Skoudy A, Ramaekers F, Real FX. Intermediate filaments as differentiation markers of normal pancreas and pancreas cancer. Am J Pathol 1992;140(3):559-68.
89. Walsh N, O'Donovan N, Kennedy S, et al. Identification of pancreatic cancer invasion-related proteins by proteomic analysis. Proteome Sci 2009;7:3.
90. Karantza V. Keratins in health and cancer: more than mere epithelial cell markers. Oncogene;2011;30(2):127-38.
91. Thompson EW, Newgreen DF, Tarin D. Carcinoma invasion and metastasis: a role for epithelial-mesenchymal transition? Cancer Res 2005;65(14):5991-5; discussion 5.
92. Wicki A, Lehembre F, Wick N, Hantusch B, Kerjaschki D, Christofori G. Tumor invasion in the absence of epithelial-mesenchymal transition: podoplanin-mediated remodeling of the actin cytoskeleton. Cancer Cell 2006;9(4):261-72.
93. De Wever O, Pauwels P, De Craene B, et al. Molecular and pathological signatures of epithelial-mesenchymal transitions at the cancer invasion front. Histochem Cell Biol 2008;130(3):481-94.
94. Nakajima S, Doi R, Toyoda E, et al. N-cadherin expression and epithelial-mesenchymal transition in pancreatic carcinoma. Clin Cancer Res 2004;10(12 Pt 1):4125-33.
95. Ikushima H, Miyazono K. TGFbeta signalling: a complex web in cancer progression. Nat Rev Cancer;2010;10(6):415-24.
96. Papageorgis P, Lambert AW, Ozturk S, et al. Smad signaling is required to maintain epigenetic silencing during breast cancer progression. Cancer Res;2010;70(3):968-78.
97. Mani SA, Yang J, Brooks M, et al. Mesenchyme Forkhead 1 (FOXC2) plays a key role in metastasis and is associated with aggressive basal-like breast cancers. Proc Natl Acad Sci U S A 2007;104(24):10069-74.
98. He X, Zheng Z, Li J, et al. DJ-1 promotes invasion and metastasis of pancreatic cancer cells by activating SRC/ERK/uPA. Carcinogenesis;2012;33(3):555-62.
99. Xia Y, Schneyer AL. The biology of activin: recent advances in structure, regulation and function. J Endocrinol 2009;202(1):1-12.
100. Kong B, Michalski CW, Erkan M, Friess H, Kleeff J. From tissue turnover to the cell of origin for pancreatic cancer. Nat Rev Gastroenterol Hepatol;2011;8(8):467-72.
101. Mott JD, Werb Z. Regulation of matrix biology by matrix metalloproteinases. Curr Opin Cell Biol 2004;16(5):558-64.
102. Behzadian MA, Wang XL, Windsor LJ, Ghaly N, Caldwell RB. TGF-beta increases retinal endothelial cell permeability by increasing MMP-9: possible role o
glial cells in endothelial barrier function. Invest Ophthalmol Vis Sci 2001;42(3):853-9.
103. Ubiali F, Nava S, Nessi V, et al. Allorecognition of human neural stem cells by peripheral blood lymphocytes despite low expression of MHC molecules: role of TGF-beta in modulating proliferation. Int Immunol 2007;19(9):1063-74.